Treatment of Fibrodysplasia Ossificans Progressiva

ABSTRACT

Methods for treating Fibrodysplasia Ossificans Progressiva (FOP) are provided. Such methods involve administering to a subject having FOP an effective regime of an activin receptor type 2A (ACVR2A) and/or an activin receptor type 2B (ACVR2B) antagonist or an activin receptor type 1 (ACVR1) antagonist. Antagonists include fusion proteins of one or more extracellular domains (ECDs) of ACVR2A, ACVR2B and/or ACVR1 and the Fc domain of an immunoglobulin heavy chain, and antibodies against ACVR2A, ACVR2B, ACVR1 or Activin A.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/850,844 filed Sep. 10, 2015, which claims the benefit under 35 USC119(e) of US Provisional Application Nos. 62/049,869 filed Sep. 12,2014, and 62/141,775 filed Apr. 1, 2015, the disclosures each of whichare herein incorporated by reference in their entireties

REFERENCE TO A SEQUENCE LISTING

The application refers to sequences written in the fileT0037US02_SEQLST.txt, created on, Aug. 28, 2017, which is 10,051 bytes.The information contained in this file is hereby incorporated byreference.

BACKGROUND

Fibrodysplasia Ossificans Progressiva (FOP) is an autosomal dominantdisorder characterized by early onset, episodic and progressiveossification of skeletal muscle and associated connective tissue. FOP isdriven by mutations in the intracellular domain of ACVR1 (ALK2), withthe great majority altering Arginine 206 to Histidine (R206H) (Pignolo,R. J. et al. 2011, Orphanet J. Rare Dis. 6:80). ACVR1 is a type Ireceptor for bone morphogenic proteins (BMPs). The R206H mutation, amongothers, is believed to increase the sensitivity of the receptor toactivation and render it more resistant to silencing. No effectivemedical therapy is known for FOP.

SUMMARY OF THE CLAIMED INVENTION

The invention provides methods of treating Fibrodysplasia OssificansProgressiva (FOP), comprising administering to a subject having FOP aneffective regime of an activin receptor type 2A (ACVR2A) and/or anactivin receptor type 2B (ACVR2B) antagonist. In some methods, theantagonist comprises an ACVR2A or ACVR2B extracellular domain. In somemethods, the antagonist comprises an ACVR2A or ACVR2B Fc fusion protein.In some methods, the isotype of the Fc fusion protein is human IgG1. Insome methods, the antagonist comprises an ACVR2A extracellular domainlinked to an ACVR2B extracellular domain. In some methods, theantagonist further comprises an Fc domain. In some methods, theantagonist comprises an ACVR2A extracellular domain fused to a first Fcdomain and an ACVR2B extracellular domain fused to a second Fc domainwherein the first and second Fc domains are complexed with one another.In some methods, the antagonist comprises a linker between the ACVR2Aand ACVR2B extracellular domains, each fused to an Fc domain. In somemethods, the antagonist is a fusion protein comprising an ACVR2Aextracellular domain, an ACVR2B extracellular domain and an Fc domain.In some methods, an effective regime of an ACVR2A antagonist and anACVR2B antagonist is administered. In some methods, the ACVR2Aantagonist is an ACVR2A Fc fusion protein and the ACVR2B antagonist isan ACVR2B Fc fusion protein. In some methods, the antagonist is anantibody to ACVR2A or ACVR2B. In some methods, the subject does not haveand is not at risk of type II diabetes, muscular dystrophy, amyotrophiclateral sclerosis (ALS) or osteoporosis.

The invention further provides methods of treating FOP, comprisingadministering to a subject having FOP an effective regime of an activinreceptor type 1 (ACVR1) antagonist. In some methods, the antagonistcomprises an ACVR1 extracellular domain. In some methods, the antagonistcomprises an ACVR1 fusion protein. In some methods, the isotype of theFc fusion protein is human IgG1. In some methods, the antagonist is anantibody to ACVR1.

The invention further provides methods of treating FibrodysplasiaOssificans Progressiva (FOP), comprising administering to a subjecthaving FOP an effective regime of an activin receptor type 2A (ACVR2A)and/or an activin receptor type 2B (ACVR2B) antagonist in combinationwith an activin receptor type 1 (ACVR1) antagonist. In some methods, theantagonist comprises an ACVR1, ACVR2A and/or ACVR2B extracellulardomain. In some methods, the antagonist comprises an ACVR1, ACVR2Aand/or ACVR2B Fc fusion protein. In some methods, the isotype of the Fcfusion protein is human IgG1. In some methods, the antagonist is anantibody to ACVR1, ACVR2A and/or ACVR2B.

The invention further provides a method of treating FibrodysplasiaOssificans Progressiva (FOP), comprising administering to a subjecthaving FOP an effective regime of an antibody against Activin A.Optionally, the antibody competes for binding with antibody comprisingthe heavy and light chain variable regions of the antibody designatedH4H10446P, H4H10430P or A1. Optionally, the antibody comprises the heavyand light chain variable regions of the antibody designated H4H10446P,H4H10430P or A1. Optionally, the antibody is a chimeric, veneered,humanized or human antibody. Optionally, the antibody is an intactantibody. Optionally, the antibody is a human kappa IgG1 antibody.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows microCT data from mice showing ectopic bone formation at 6weeks post initiation of tamoxifen administration with and withoutACVR2A-Fc/ACVR2B-Fc treatment. Nine out of ten control mFc treated mice(numbers 1 to 9) showed ectopic bone formation at 6 weeks post tamoxifenadministration. Most common locations are hind limb, neck region andsternum. Two out of eleven ACVR2A-Fc/ACVR2B-Fc treated mice (numbers 12and 14) showed ectopic bone formation at 6 weeks post tamoxifenadministration. The ectopic bone lesions in these two mice were of smallsize compared to those seen in the mFc treated group and both located atthe sternum.

FIG. 2 shows microCT data from mice showing ectopic bone formation 4weeks post initiation of tamoxifen administration with or withoutLDN212854 treatment. Numbers 1 to 8 correspond to tamoxifen+vehicletreated mice. Large ectopic bone nodules have formed in mice numbered 1,2, 3, 4, 5 and 7, and small ectopic bone nodules have formed in micenumbered 6 and 8. Numbers 9-16 correspond to tamoxifen+LDN212854 treatedmice. Small ectopic bone nodules have formed in mice numbered 9, 12 and13. No ectopic bone nodules could be detected in mice numbered 10, 11,14, 15 or 16.

FIG. 3 shows microCT data for mice disposed to ectopic bone formationtreated with an antibody against Activin A, an isotype matchedirrelevant control antibody, and ACVR2A-Fc. The antibody against ActivinA inhibited formation of ectopic bone nodules most effectively.

FIG. 4 shows microCT data for mice disposed to ectopic bone formationtreated with an antibody against Activin A, an isotype matchedirrelevant control antibody, and an antibody against Acvr2a/Acvr2b. Theantibody against Activin A and the antibody against Acvr2a/Acvr2binhibited formation of ectopic bone nodules.

FIG. 5 shows microCT data for mice disposed to ectopic bone formationtreated with varying doses of an antibody against Activin A comparedwith an isotype matched irrelevant control antibody. Dosages between 1mg/kg and 25 mg/kg were shown to be effective with 10 mg/kg being themost effective dose tested.

DEFINITIONS

Antagonists are typically provided in isolated form. This means that anantagonist is typically at least 50% w/w pure of interfering proteinsand other contaminants arising from its production or purification, butdoes not exclude the possibility that the antagonist is combined with anexcess of pharmaceutical acceptable carrier(s) or other vehicle intendedto facilitate its use. Sometimes antagonists are at least 60, 70, 80,90, 95 or 99% w/w pure of interfering proteins and contaminants fromproduction or purification.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Non-conservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention for a variableregion or EU numbering for a constant region. For other proteins,sequence identity can be determined by aligning sequences usingalgorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), using default gap parameters, or by inspection, and thebest alignment. After alignment, if a subject antibody region (e.g., theentire mature variable region of a heavy or light chain) is beingcompared with the same region of a reference antibody, the percentagesequence identity between the subject and reference antibody regions isthe number of positions occupied by the same amino acid in both thesubject and reference antibody region divided by the total number ofaligned positions of the two regions, with gaps not counted, multipliedby 100 to convert to percentage.

Compositions or methods “comprising” one or more recited elements caninclude other elements not specifically recited. For example, acomposition that comprises antibody can contain the antibody alone or incombination with other ingredients.

A humanized antibody is a genetically engineered antibody in which theCDRs from a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No.6,407,213; Adair, U.S. Pat. Nos. 5,859,205 and 6,881,557; Foote, U.S.Pat. No. 6,881,557). The acceptor antibody sequences can be, forexample, a mature human antibody sequence, a composite of suchsequences, a consensus sequence of human antibody sequences, or agermline region sequence. Thus, a humanized antibody is an antibodyhaving some or all CDRs entirely or substantially from a donor antibodyand variable region framework sequences and constant regions, ifpresent, entirely or substantially from human antibody sequences.Similarly, a humanized heavy chain has at least one, two and usually allthree CDRs entirely or substantially from a donor antibody heavy chain,and a heavy chain variable region framework sequence and heavy chainconstant region, if present, substantially from human heavy chainvariable region framework and constant region sequences. Similarly, ahumanized light chain has at least one, two and usually all three CDRsentirely or substantially from a donor antibody light chain, and a lightchain variable region framework sequence and light chain constantregion, if present, substantially from human light chain variable regionframework and constant region sequences. Other than nanobodies and dAbs,a humanized antibody comprises a humanized heavy chain and a humanizedlight chain. A CDR in a humanized antibody is substantially from acorresponding CDR in a non-human antibody when at least 85%, 90%, 95% or100% of corresponding residues (as defined by Kabat) are identicalbetween the respective CDRs. The variable region framework sequences ofan antibody chain or the constant region of an antibody chain aresubstantially from a human variable region framework sequence or humanconstant region, respectively, when at least 85, 90, 95 or 100% ofcorresponding residues defined by Kabat are identical.

Although humanized antibodies often incorporate all six CDRs (preferablyas defined by Kabat) from a mouse antibody, they can also be made withless than all CDRs (e.g., at least 3, 4, or 5 CDRs from a mouseantibody) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos etal., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al.,Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology,164:1432-1441, 2000).

A chimeric antibody is an antibody in which the mature variable regionsof light and heavy chains of a non-human antibody (e.g., a mouse) arecombined with human light and heavy chain constant regions. Suchantibodies substantially or entirely retain the binding specificity ofthe mouse antibody, and are about two-thirds human sequence.

A veneered antibody is a type of humanized antibody that retains someand usually all of the CDRs and some of the non-human variable regionframework residues of a non-human antibody, but replaces other variableregion framework residues that can contribute to B- or T-cell epitopes,for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) withresidues from the corresponding positions of a human antibody sequence.The result is an antibody in which the CDRs are entirely orsubstantially from a non-human antibody and the variable regionframeworks of the non-human antibody are made more human-like by thesubstitutions.

A human antibody can be isolated from a human, or otherwise result fromexpression of human immunoglobulin genes (e.g., in a transgenic mouse,in vitro or by phage display). Methods for producing human antibodiesinclude the trioma method of Oestberg et al., Cys muoma 2:361-367(1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S.Pat. No. 4,634,666. The monoclonal antibodies can also be produced bytransgenic mice bearing human immune system genes, such as theVeloclmmune® mouse from Regeneron Pharmaceuticals, Inc. (Murphy, PNAS111 no. 14, 5153-5158 (2014), Xenomouse, Jakobovits, NatureBiotechnology 25, 1134-1143 (2007) or HuMAb mouse from Medarex, Inc.(Lonberg, Handbook Exp. Pharmacol. 181, 69-97 (2008); Lonberg et al.,WO93/12227 (1993); U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299,U.S. Pat. No. 5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No.5,770,429, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat.No. 5,625,126, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996),Kucherlapati, WO 91/10741 (1991). Human antibodies can also be producedby phage display methods (see, e.g., Dower et al., WO 91/17271 andMcCafferty et al., WO 92/01047, U.S. Pat. No. 5,877,218, U.S. Pat. No.5,871,907, U.S. Pat. No. 5,858,657, U.S. Pat. No. 5,837,242, U.S. Pat.No. 5,733,743 and U.S. Pat. No. 5,565,332).

When an antagonist is said to retain a property of a parental antibodyfrom which it was derived, the retention can be complete or partial.Complete retention of an activity means the activity of the antagonistis the same within experimental error or greater than that of themolecule from which it was derived. Partial retention of activity meansactivity significantly above background level of a negative control(i.e., beyond experimental error) and preferably at least 50% of thecorresponding activity of the molecule from which it was derived.

Two antibodies have the same epitope if all amino acid mutations in theantigen that reduce or eliminate binding of one antibody reduce oreliminate binding of the other. Two antibodies have overlapping epitopesif some amino acid mutations that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50%, but preferably 75%,90% or 99%, as measured in a competitive binding assay. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.

DETAILED DESCRIPTION I. Overview

Methods for treating Fibrodysplasia Ossificans Progressiva (FOP) areprovided. Such methods involve administering to a subject having FOP aneffective regime of an activin receptor type 2A (ACVR2A) and/or anactivin receptor type 2B (ACVR2B) antagonist and/or an activin receptortype 1 (ACVR1) antagonist, and/or an Activin A antagonist. Suchantagonists include fusion proteins comprising one or more extracellulardomains (ECDs) of ACVR2A, ACVR2B and/or ACVR1 and the Fc domain of animmunoglobulin heavy chain. Antibody antagonists of ACVR2A, ACVR2B,ACVR1 or Activin A are also provided.

II. ACVR1, ACVR2A, ACVR2B and Activin A

The transforming growth factor β (TGFβ) superfamily of ligands includes,for example, bone morphogenetic proteins (BMPs) and growth anddifferentiation factors (GDFs). The receptors for these ligands areheteromeric receptor complexes made up of type I and type IItransmembrane serine/threonine kinase receptors. Examples of type Ireceptors include activin receptor type IA (ACTRIA, ACVR1, or ALK2), BMPreceptor type IA and BMP receptor type IB. Examples of type II receptorsinclude activin receptors type IIA and IIB (ACTRIIA or ACVR2A andACTRIIB or ACVR2B) and BMP receptor type II. The ligands of the TGFβsuperfamily each have differing affinities for the different type I andtype II receptors.

Both the type I and type II receptors have an extracellular ligandbinding domain (ECD) and an intracellular serine/threonine kinasedomain. In addition, the type I receptors have a glycine/serine-richregion (GS-box) preceding the kinase domain and a L45 loop within thekinase domain. Both receptors work together for ligands to activatedownstream signaling pathways, such as Smad and non-Smad signalingpathways. Activation involves ligand binding, ligand-receptoroligomerization and transphosphorylation of the GS box of the type Ireceptor by the type II receptor kinase. The type II receptor kinase isconstitutively active and has a role in ligand binding and activation ofthe type I receptor.

ACVR1, also known as activin a receptor type I, ACVR1A, ACVRLK2, orALK2, is a type I receptor for the TGFβ superfamily of ligands. ACVR1has serine/threonine kinase activity and phosphorylates Smad proteinsand activates downstream signaling pathways. ACVR1 is found in manytissues of the body including skeletal muscle and cartilage and helps tocontrol the growth and development of the bones and muscles. Asdescribed elsewhere herein, certain mutations in the ACVR1 gene causeFOP. Examples of ACVR1 activity include the ability to bind to ligands,the ability to form a complex with a type II receptor, or the ability toactivate downstream signaling pathways, such as the Smad pathway.

ACVR2, also known as activin receptor type II, is a type II receptor forthe TGFβ superfamily of ligands. There are at least two ACVR2 receptors,for example, activin receptor type IIA (ACVR2A or ACTRIIA) and activinreceptor type IIB (ACVR2B or ACTRIIB). Reference to ACVR2 includeseither or both of ACVR2A and ACVR2B. ACVR2A and ACVR2B can be expressedin multiple tissues, including skeletal muscle, stomach, heart,endometrium, testes, prostate, ovary, and neural tissues.

On ligand binding, an ACVR2 receptor forms a complex with a type Ireceptor, such as ACVR1, and phosphorylates the GS box of the type Ireceptor, thus enhancing the kinase activity of the type I receptor.Examples of ACVR2A and ACVR2B activity include the ability to bind toligands, the ability to form a complex with a type I receptor, or theability to phosphorylate a type I receptor.

An exemplary form of human ACVR2A has Swiss Prot accession numberP27037. Residues 1-19 are a signal peptide, residues 20-135 are anextracellular domain, residues 59-116 are an activin types I and IIreceptor domain, residues 136-161 are a transmembrane domain andresidues 162-513 are a cytoplasmic domain. An exemplary form of humanACVR2B is assigned Swiss Prot Number Q13705. Residues 1-18 are a signalsequence, residues 19-137 are an extracellular domain, residues 27-117are an activin types I and II receptor domain, residues 138-158 are atransmembrane domain and residues 159-512 are a cytoplasmic domain. Anexemplary form of human ACVR1 has Swiss Prot accession number Q04771.Residues 1-20 are a signal sequence, residues 21-123 are extracellulardomain, residues 33-104 are an activin types I and II receptor domain,residues 124-146 are a transmembrane domain and residues 147-509 are acytoplasmic domain. Reference to any of ACVR1, ACVR2A and ACVR2Bincludes these exemplary forms, known isoforms and polymorphismsthereof, such as those listed in the Swiss Prot database, cognate formsfrom other species, and other variants having at least 90, 95, 96, 97,98 or 99% sequence identity with an exemplified form.

Residues of forms of ACVR2A, ACVR2B and ACVR1 other than the exemplifiedsequences defined above are numbered by maximum alignment with thecorresponding exemplified sequences so aligned residues are allocatedthe same number. Substitutions from exemplified sequences can beconservative or non-conservative substitutions. Reference to ACVR1,ACVR2A or ACVR2B also includes intact extracellular domains (e.g.,residues 20-135, 19-137 or 21-123 of ACVR2A, ACVR2B and ACVR1,respectively) or a portion thereof free or substantially free oftransmembrane and cytoplasmic portion. Portions of an extracellulardomain retain sufficient residues of the intact extracellular domain tobind at least one ligand or counter receptor that binds to the intactextracellular domain and thereby antagonize the relevant receptor (e.g.,residues 59-116, 27-117 or 33-104 of ACVR2A, ACVR2B and ACVR1,respectively).

Activin A in humans can exist as a homo or heterodimeric protein. Thehomodimeric protein contains a homodimeric beta A subunit pair. Theheterodimeric protein contains a beta subunit and a beta B, beta C orbeta E subunit (i.e., beta A beta B, beta A beta C, or beta A beta E.The subunits are each expressed as precursor polypeptides including asignal peptide, propeptide and mature polypeptide. An exemplary form ofhuman beta A subunit precursor is a polypeptide of length 426 aminoacids designated Swiss Prot P08476 of which residues 1-20 are a signalpeptide, residues 21-310 are a propeptide and residues 311-426 are themature polypeptide. An exemplary form of a beta B subunit precursorpolypeptide is designated Swiss Prot P09529 of which residues 1-28 are asignal peptide, residues 29-292 a propeptide and residues 293-407 amature polypeptide. An exemplary form of a beta C subunit is designatedSwiss Prot P55103, of which residues 1-18 are a signal peptide, residues19-236 are a propeptide and residues 237-352 are a mature polypeptide.An exemplary form of a beta E subunit precursor is designated Swiss ProtP58166 of which residues 1-19 are a signal peptide, residues 20-236 area propeptide and residues 237-350 are a mature polypeptide. Severalvariants of these sequences are known as described in the Swiss ProtData base. Reference to Activin A includes any of the beta A homodimer,beta A beta B, beta A beta C and beta A beta E heterodimer forms, aswell as their subunits, as well as their precursors in which subunitsare attached to the propeptide and/or signal peptide defined by theexemplary Swiss Prot sequences provided or other natural occurring humanforms of these sequences. Activin A signals through binding to ACVR2A orACVR2B, but is not known to be a ligand for ACVR1.

III. Antagonists of ACVR1, ACVR2A, ACVR2B

Antagonists of the type I receptor ACVR1 and of the type II receptorACVR2 proteins (e.g., ACVR2A and/or ACVR2B) are provided for treatingFOP. Such antagonists can antagonize receptors directly by binding tothe receptor (as for an antibody to ACVR1, ACVR2A or ACVR2B) orindirectly by binding to a ligand or counter receptor and inhibiting theligand or counter receptor from binding to ACVR1, ACVR2A or ACVR2B (asfor a fusion protein of ACVR1, ACVR2A or ACVR2B) among other mechanisms.Antagonists of ACVR2A and ACVR2B can also bind to Activin A.

An ACVR1, ACVR2A or ACVR2B antagonist provided herein can inhibit orreduce the activity of ACVR1, ACVR2A and/or ACVR2B by at least 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more relativeto a control cell or animal model that did not receive the antagonist.

Any antagonist of ACVR1, ACVR2A or ACVR2B can be used in the methods fortreating FOP. The antagonist can comprise, for example, an ACVR1, ACVR2Aor ACVR2B polypeptide, such as an extracellular domain, an antagonistantibody, or a small molecule inhibitor.

A. Extracellular Domains of ACVR1, ACVR2A and ACVR2B Polypeptides

Antagonists include ACVR1, ACVR2A and ACVR2B proteins and fragmentsthereof effective to inhibit at least one activity of ACVR1, ACVR2A andACVR2B, respectively. Such antagonists typically include theextracellular domain of ACVR1, ACVR2A or ACVR2B or a portion thereof.Preferably, such extracellular domains are entirely or substantiallyfree of the transmembrane and cytoplasmic regions (i.e., any remainingresidues from these regions have no significant effect on function ofthe extracellular domain). In other words, the ACVR2A, ACVR2B or ACVR1component of such antagonists consists of or consists essentially of theentire extracellular domain of ACVR2A, ACVR2B or ACVR1 ora portionthereof as defined above Such antagonists may or may not include othercomponent(s) distinct from ACVR2A, ACVR2B or ACVR1 as further describedbelow. Such extracellular domains free or substantially free oftransmembrane and cytoplasmic domains are soluble. Such extracellulardomains can function as an antagonist by binding to a soluble ligand orcounter receptor, effectively competing with the ligand or counterreceptor binding to the ACVR1, ACVR2A or ACVR2B cell surface receptor,thereby modulating (reducing) the availability of the ligand or counterreceptor in vivo.

Soluble extracellular domains can be initially expressed with a signalsequence, which is cleaved in the course of expression. The signalsequence can be a native signal sequence of an ACVR1, ACVR2A or ACVR2B,such as those described in U.S. Pat. No. 7,709,605, which isincorporated by reference herein in its entirety, or can be a signalsequence from a different protein such honey bee melittin (HBM) ortissue plasminogen activator (TPA). Alternatively, soluble extracellularACVR1, ACVR2A or ACVR2B polypeptides can be synthesized or expressedwithout a signal sequence.

The ECDs or ligand binding domains of ACVR1, ACVR2A and ACVR2B arehighly conserved among species including mouse and human. The ECDscontain a cysteine rich region and a C-terminal tail region. The ECDs ofACVR1, ACVR2A and ACVR2B bind to a diverse group of TGFβ family ligands,including, for example, Activin A, myostatin (GDF-8), GDF-11 and BMPs.See, e.g., Souza et al. (2008) Molecular Endocrinology 22(12):2689-2702.

Examples of ACVR2A and ACVR2B polypeptides and soluble ACVR2A and ACVR2Bpolypeptides include those disclosed in U.S. Pat. No. 7,842,633; U.S.Pat. No. 7,960,343; and U.S. Pat. No. 7,709,605, each of which isincorporated by reference herein in their entirety.

The ECD of an ACVR1, ACVR2A or ACVR2B polypeptide can be mutated suchthat the variant polypeptide has altered ligand binding properties(e.g., binding specificity or affinity). Some variant ACVR1, ACVR2A orACVR2B polypeptides have altered binding affinity (e.g., elevated orreduced) for a specific ligand. Variants have at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the naturallyoccurring ACVR1, ACVR2A or ACVR2B sequences, and retain biologicalactivity and hence have an ACVR1, ACVR2A or ACVR2B activity as describedelsewhere herein. Active variants and fragments of ACVR2A and ACVR2B aredescribed, for example, in U.S. Pat. No. 7,842,633; U.S. Pat. No.7,960,343; and U.S. Pat. No. 7,709,605, each of which is incorporated byreference herein in its entirety.

Assays to measure ACVR1, ACVR2A or ACVR2B activity are disclosed ine.g., U.S. Pat. No. 7,842,633; U.S. Pat. No. 7,960,343; and U.S. Pat.No. 7,709,605. For example, an ACVR1, ACVR2A or ACVR2B polypeptidevariant can be screened for the ability to bind a ligand or for theability to prevent binding of a ligand to an ACVR1, ACVR2A or ACVR2Breceptor protein.

B. Fusion Proteins

The ACVR1, ACVR2A and ACVR2B polypeptides described above can beexpressed as fusion proteins having at least a portion of an ACVR1,ACVR2A and/or ACVR2B polypeptide and one or more fusion domains.

Fusion domains include an immunoglobulin heavy chain constant region(Fc), human serum albumin (HSA), glutathione S transferase (GST),protein A, protein G, or any fusion domain which can be useful instabilizing, solubilizing, isolating or multimerizing a fusion protein.

An Fc domain of an immunoglobulin heavy chain is a preferred domain forfusion proteins. Fusions with the Fc portion of an immunoglobulin conferdesirable pharmacokinetic properties on a wide range of proteins (e.g.,increases stability and/or serum half-life of the protein). Thus, theinvention provides fusion proteins comprising at least one ECD of anACVR1, ACVR2A and/or ACVR2B fused to an Fc domain of an immunoglobulin.

The Fc domain for use in the present methods can be from anyimmunoglobulin. Any of the various classes of immunoglobulin can beused, including IgG, IgA, IgM, IgD and IgE. Within the IgG class thereare different subclasses or isotypes, including, for example, IgG₁,IgG₂, IgG₃ and IgG₄. In one embodiment, the Fc fusion protein comprisesthe Fc domain of an IgG molecule. In a further embodiment, the Fc domainis from an IgG₁ molecule. The immunoglobulin molecule can be of anyanimal type, including, for example, a mammal, a rodent, a human, amouse, a rat, a hamster or a rabbit. In one embodiment, theimmunoglobulin Fc domain is from a mammal. In another embodiment, the Fcdomain is from a human. In yet another embodiment, the Fc domain is froma rodent, such as a mouse or rat. In a specific embodiment, the Fcdomain of the fusion protein is from human IgG1.

The Fc-fusion proteins provided herein can be made by any method knownin the art. The Fc-fusion proteins can include at least CH2 and CH3regions, and typically at least a portion of a hinge region. Althoughthe CH1 region can be present, it is typically omitted in fusionproteins.

The fusion can be made at any site within the Fc portion of animmunoglobulin constant domain. Fusions can be made to the C-terminus ofthe Fc portion of a constant domain, or immediately N-terminal to theCH1 region of the heavy chain. Particular sites can be selected tooptimize the biological activity, secretion or binding characteristicsof the Fc-fusion protein.

In some cases, a nucleic acid encoding the ECD of ACVR1, ACVR2A and/orACVR2B is fused C-terminally to a nucleic acid encoding the N-terminusof an immunoglobulin constant domain sequence. In other cases,N-terminal fusions are also possible. It is also possible to fuse an ECDof ACVR1, ACVR2A and/or ACVR2B to both the N-terminus and the C-terminusof an immunoglobulin constant domain sequence.

For the production of immunoglobulin fusions, see also U.S. Pat. No.5,428,130, U.S. Pat. No. 5,843,725, U.S. Pat. No. 6,018,026 andWO2005/070966, each of which is incorporated by reference herein intheir entirety.

A fusion protein can be produced, for example, by recombinant expressionof a nucleic acid encoding the fusion protein. For example, the fusionprotein can be made by fusing a nucleic acid encoding an ECD of ACVR1,ACVR2A and/or ACVR2B to a nucleic acid encoding an Fc domain. The ACVR1,ACVR2A and/or ACVR2B ECD nucleic acid can be fused to the N-terminus ofa nucleic acid encoding an Fc domain or can be fused to the C-terminusof a gene encoding an Fc domain. Alternatively, the ECD can be fused atany position in the Fc domain.

The ECD fusion proteins can also include a linker. In the case of an Fcfusion protein, the linker can be positioned between the ACVR1, ACVR2Aor ACVR2B ECD and the Fc domain, optionally replacing part or all of thehinge region. The linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,30, 50 or more amino acids that are relatively free of secondarystructure. A linker can be rich in glycine and proline residues and can,for example, contain repeating sequences of threonine/serine andglycines (e.g., TG₄ or SG₄ repeats).

Two or more ECD-Fc fusion proteins can be joined together by a linker.In such cases, the linker can be positioned between the ECDs or thelinker can be positioned between the Fc domains to join the fusionproteins together. For example, 1, 2, 3, 4 or more ACVR1, ACVR2A and/orACVR2B Fc fusion proteins can be linked together.

Examples of ACVR2A and/or ACVR2B ECD fusion proteins have beendescribed, such as those disclosed in U.S. Pat. No. 7,842,633; U.S. Pat.No. 7,960,343; and U.S. Pat. No. 7,709,605, each of which isincorporated by reference herein in their entirety.

One example of an ACVR2A antagonist is known as Sotatercept (also calledACE-011). Sotatercept contains the ECD of ACVR2A fused to a human IgG1Fc domain and is described in detail in Carrancio et al., (2014) BritishJ Haematology. 165(6):870-872, which is incorporated by reference hereinin its entirety.

One example of an ACVR2B antagonist is known as ACE-031. ACE-031contains the ECD of ACVR2B fused to a human IgG1 Fc domain and isdescribed in detail in Sako et al., (2010) J. Biol. Chem.285(27):21037-21048, which is incorporated by reference herein in itsentirety.

Examples of ACVR1 ECD fusion proteins are known, such as those disclosedin Berasi, et al., (2011) Growth Factors, 29(4):128-139; which isincorporated by reference herein in its entirety.

C. Hybrid ECD Fusion Proteins

Hybrid or multispecific ECD fusion protein antagonists are alsoprovided. Hybrid ECD fusion proteins can comprise a combination of twoor more ACVR1, ACVR2A and/or ACVR2B ECDs. For example, the fusionproteins can comprise 1, 2, 3, 4 or more molecules of an ACVR1, ACVR2Aand/or ACVR2B ECD. In one embodiment, the antagonist comprises an ACVR2AECD linked to an ACVR2B ECD. In a further embodiment, the antagonistfurther comprises an Fc domain.

In one embodiment, a fusion protein can comprise one or more moleculesof an ACVR2A ECD and one or more molecules of an ACVR2B ECD. In anotherembodiment, a fusion protein can comprise one or more molecules of anACVR1 ECD and one or more molecules of an ACVR2A ECD. In anotherembodiment, a fusion protein can comprise one or more molecules of anACVR1 ECD and one or more molecules of an ACVR2B ECD.

In one embodiment, a fusion protein comprises one or more ACVR2A ECD-Fcfusion proteins and one or more ACVR2B ECD-Fc fusion proteins which arecomplexed together. In another embodiment, a fusion protein comprisesone or more ACVR1 ECD-Fc fusion proteins and one or more ACVR2A ECD-Fcfusion proteins which are complexed together. In another embodiment, afusion protein comprises one or more ACVR1 ECD-Fc fusion proteins andone or more ACVR2B ECD-Fc fusion proteins which are complexed together.In such cases, the fusion proteins can be joined together via their Fcdomains, for example, by at least one disulfide linkage or by a linkersequence. Alternatively, the ECD portions of the fusion protein can bejoined together by a linker sequence.

In one embodiment, the antagonist comprises an ACVR2A ECD fused to afirst Fc domain and an ACVR2B ECD fused to a second Fc domain. In suchcases, the Fc domains can be complexed with one another. In anotherembodiment, the antagonist comprises a linker between the ACVR2A andACVR2B ECDs, each fused to an Fc domain.

The fusion proteins can be constructed to generate ACVR1, ACVR2A, and/orACVR2B antagonists in a tandem format. In one embodiment, a fusionprotein comprises two or more ECDs from ACVR1, ACVR2A and/or ACVR2B intandem followed by an Fc domain. In some cases the ECDs arranged intandem are separated by a linker sequence. Such a tandem fusion proteincan comprise 1, 2, 3, 4 or more ACVR1, ACVR2A and/or ACVR2B ECDs.

D. Antibody Antagonists

An ACVR1, ACVR2A or ACVR2B antagonist includes antibodies against (inother words specifically binding to) any of these receptors, preferablyantibodies having an epitope within the extracellular domain. Specificbinding of an antibody or fusion protein to its target antigen means anaffinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Specific bindingis detectably higher in magnitude and distinguishable from non-specificbinding occurring to at least one unrelated target. Methods forpreparing antibodies are known to the art. See, for example, Kohler &Milstein (1975) Nature 256:495-497; and Harlow & Lane (1988) Antibodies:a Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.

Any antibody that inhibits or reduces the activity of ACVR1, ACVR2Aand/or ACVR2B (e.g., an antagonist antibody) can be used. Such ACVR2Aand ACVR2B antibodies include, for example, those antibodies disclosedin U.S. Pat. No. 8,486,403, U.S. Pat. No. 8,128,933, WO2009/137075, andLach-Trifilieff, et al. (2014) Mol. Cell Biol. 34(4):606-618, each ofwhich is incorporated by reference herein in their entirety. Humanized,chimeric and veneered forms of any of these antibodies are included asare antibodies competing for binding therewith.

In one embodiment, the antibody is an anti-ACVR2A antibody. In anotherembodiment, the antibody is an anti-ACVR2B antibody. In otherembodiments, the antibody can be a bispecific antibody against bothACVR2A and ACVR2B. In another embodiment, the antibody is an anti-ACVR1antibody. In other embodiments, the antibody can be a bispecificantibody against both ACVR1 and ACVR2A or against both ACVR1 and ACVR2B.

The term “antibody” covers intact antibodies with two pairs of heavy andlight chains, antibody fragments that can bind antigen (e.g., Fab,F(ab′)₂, Fv, single chain antibodies, diabodies, antibody chimeras,hybrid antibodies, bispecific antibodies, humanized antibodies, and thelike), and recombinant peptides comprising the forgoing.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include Fab, F(ab′)2, and Fvfragments; diabodies; linear antibodies (Zapata et al. (1995) ProteinEng. 10:1057-1062); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The antibody can be monoclonal or polyclonal. A “monoclonal antibody” isan antibody obtained from a population of substantially homogeneousantibodies, that is, the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that canbe present in minor amounts. Monoclonal antibodies are often highlyspecific, being directed against a single antigenic site. Furthermore,in contrast to conventional (polyclonal) antibody preparations thattypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is typically directedagainst a single determinant on the antigen. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, such as thoseproduced by a clonal population of B-cells, and does not requireproduction of the antibody by any particular method.

Monoclonal antibodies to be used in accordance with the methods providedherein can be made by the hybridoma method first described by Kohler etal. (1975) Nature 256:495, or a modification thereof. Typically, ananimal, such as a mouse, is immunized with a solution containing anantigen (e.g., an ACVR1, ACVR2A and/or ACVR2B polypeptide, or Activin Aparticularly the extracellular domain (in receptors) or a portionthereof).

Immunization can be performed by mixing or emulsifying theantigen-containing solution in saline, preferably in an adjuvant such asFreund's complete adjuvant, and injecting the mixture or emulsionparenterally. After immunization of the animal, the spleen (andoptionally, several large lymph nodes) are removed and dissociated intosingle cells. The spleen cells can be screened by applying a cellsuspension to a plate or well coated with the antigen of interest. TheB-cells expressing membrane bound immunoglobulin specific for theantigen bind to the plate and are not rinsed away. Resulting B-cells, orall dissociated spleen cells, are then induced to fuse with myelomacells to form hybridomas, and are cultured in a selective medium. Theresulting cells are plated by serial dilution and are assayed for theproduction of antibodies that specifically bind the antigen of interest(and that do not bind to unrelated antigens). The selected monoclonalantibody (mAb)-secreting hybridomas are then cultured either in vitro(e.g., in tissue culture bottles or hollow fiber reactors), or in vivo(as ascites in mice).

Alternatively, the monoclonal antibodies can be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodiescan also be isolated from phage antibody libraries using the techniquesdescribed in, for example, Clackson et al. (1991) Nature 352:624-628;Marks et al. (1991) J. Mol. Biol. 222:581-597; and U.S. Pat. No.5,514,548.

“Antibodies” include chimeric, veneered, humanized and human monoclonalantibodies against any of ACVR1, ACVR2A, ACVR2B and Activin A as definedabove.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The present monoclonal antibodies or Fc fusion proteins can be any ofthe various antibody classes. In one embodiment, the monoclonal antibodyis an IgG class antibody. In other embodiments, the monoclonal antibodycan be of the IgM, IgE, IgD, or IgA class. In specific embodiments, theantibody is an isotype of IgG, such as, IgG1, IgG2, IgG3 or IgG4,particularly human IgG1, IgG2, IgG3 or IgG4.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as a C-terminal lysine of the heavy chain, canbe missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position250 and/or a Leu at position 428 (EU numbering) for increasing thehalf-life of an antibody. Substitution at any of positions 234, 235, 236and/or 237 reduces affinity for Fcγ receptors, particularly FcγRIreceptor (see, e.g., U.S. Pat. No. 6,624,821). Optionally, positions234, 236 and/or 237 in human IgG2 are substituted with alanine andposition 235 with glutamine. (See, e.g., U.S. Pat. No. 5,624,821).Effector functions can also be reduced by substitution of EFLG atpositions 232-236 with PVA (see WO14/121087). Optionally, S at position428 can be replaced by P, particularly in human IgG4 to reduce exchangebetween endogenous and exogenous immunoglobulins. Other variations canadd or remove sites of post-translational modification, such as N-linkedglycosylation at N-X-S/T motifs. Variations can also includeintroduction of knobs (i.e., replacement of one or more amino acids withlarger amino acids) or holes (i.e., replacement of one or more aminoacids with smaller amino acids) to promote formation of heterodimersbetween different heavy chains for production of bispecific antibodies.Exemplary substitutions to form a knob and hole pair are T336Y andY407T, respectively (Ridgeway et al., Protein Engineering vol. 9 no. 7pp. 617-621, 1996). Variations can also include mutations that reduceprotein A interaction (e.g., H435R and Y436F) in the EU numberingsystem. Bispecific antibodies in which one heavy chain has such avariation, and another does not, can be separated from their parentalantibodies by protein-A affinity chromatography.

Antibodies can also include antibodies specifically binding to ActivinA. Such antibodies can specifically bind to any or all of the beta Abeta A, beta A beta B, beta A beta C and beta A beta E forms of ActivinA. Some antibodies specifically bind to only one of these forms (i.e.,beta A beta A, beta A beta B, beta A beta C or beta A beta E).Specificity for the beta A beta B, beta A beta C and beta A beta E formscan be conferred by an epitope within the beta B, beta C or beta Esubunit, respectively, or for an epitope to which both components of theheterodimer contribute. Specificity for beta A beta can be conferred byan epitope contributed by both molecules within the homodimer (e.g., atthe interface of subunits). Some antibodies specifically bind to all ofthese forms of Activin A, in which case the epitope is typically on thebeta A subunit. Antibodies typically have epitopes within the maturepolypeptide component of the precursor proteins. Some antibodiesspecifically bind to any or all forms of Activin A without binding tohuman inhibin, which exists in the form of alpha (Swiss Prot P05111)beta A or alpha beta B heterodimers. Some antibodies specifically bindto any or all forms of Activin A and bind to either or both forms ofhuman inhibin. Although it is believed that such antibodies inhibitsignal transduction of Activin A through one or more of itscounterreceptors, ACVR2A and/or ACVR2B and/or BMPR2, an understanding ofmechanism is not required for use of such antibodies in methods oftreating FOP.

A substantial number of antibodies against Activin A have been reported.For example, US2015/00373339 discloses human antibodies designatedH4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P, H4H10432P2,H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2, H4H10442P2,H4H10445P2, H4H10446P2, H4H10447P2, H4H10447P2, H4H10448P2, H4H10452P2.U.S. Pat. No. 8,309,082 discloses human antibodies A1-A14. Mouseantibodies against Activin A are available from several commercialsuppliers, such as MAB3381 from R&D Systems or 9H16 from NovusBiologicals or MM0074-7L18 (a b89307) AbCam.

Preferred antibodies have an affinity for Activin A (measured at 25° C.as in Example 3 of US2015/00373339) of at least 10⁸ M⁻¹, 10⁹ M⁻¹,10¹⁰M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹, or 10¹³ M⁻¹. Some antibodies have anaffinity within a range of 10⁹-10¹² M⁻¹. Preferred antibodies inhibitsignal transduction of Activin A with an IC50 of less than 4 nM, andpreferably less than 400 pM or 40 pM. Some antibodies inhibit signaltransduction with and IC50 in a range of 4 nM to 10 pM or 3.5 nM to 35pM.

Signal transduction inhibition can be measured as in Example 6 ofUS20150037339, which is summarized as follows. A human A204rhabdomyosarcoma cell line is transfected with a Smad 2/3-luciferasereporter plasmid to produce the A204/CAGAx12-Luc cell line.A204/CAGAx12-Luc cells were maintained in McCoy's 5A supplemented with10% fetal bovine serum, penicillin/streptomycin/glutamine and 250 μg/mLof G418. For the bioassay, A204/CAGAx12-Luc cells were seeded onto96-well assay plates at 10,000 cells/well in low serum media, 0.5% FBSand OPTIMEM, and incubated at 37° C. and 5% CO₂ overnight. Activin A isserially diluted at 1:3 from 100 to 0.002 nM and added to cells startingalong with a control containing no Activin. Antibodies are seriallydiluted at 1:3 starting from 100 to 0.002 nM, 1000 to 0.02 nM, or 300 to0.005 nM including control samples containing either an appropriateisotype control antibody or no antibody and added to cells with aconstant concentration of 100 pM Activin A.

Some antibodies inhibit binding of Activin A to ACVR2A and/or ACVR2Band/or BMPR2 by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, as measured when the receptor is expressed from a cell orthe extracellular domain is fused with an Fc domain as a fusion protein,and the fusion protein is immobilized to support (e.g., a Biacore sensorchip). In such measurements, the antibody and Activin A should bepresent in equimolar amounts and the receptor or extracellular domain inexcess.

Some antibodies bind to an epitope within residues 321-343 or 391-421 offull-length Activin A, which correspond to C11-S33 and C81-E111 of themature protein.

An exemplary antibody used in the present examples is designatedH4H10446P in US2015037339. Its heavy chain variable region and heavychain CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ IDNOs:162, 164, 166 and 168, respectively, of US2015/00373339 (present SEQID NOs:1-4, respectively). Its light chain variable region and lightchain CDRs, CDRL1, CDRL2 and CDRL3 having the amino acid sequences ofSEQ ID NO:146, 148, 150 and 152, respectively, of US2015/0037339(present SEQ ID NOs:5-8, respectively). H4H10446P inhibits Activin Amediated signaling through ACVR2A and/or ACVRIIB, but does not inhibitstrongly, if at all, Activin A binding to ACRIIA or ACVR2B. Otherantibodies competing with H4H10446P for binding to human Activin A orbinding to the same epitope on human Activin A as H4H10446P are includedand sharing its inhibition of signaling are also included.

Another exemplary antibody for use in the present methods is designatedH4H10430P in US2015037339. Its heavy chain variable region and heavychain CDRs CDRH1, CDRH2 and CDRH3 having the amino acid sequences of SEQID NOs:66, 68, 70 and 72, respectively, in US2015/00373339 (present SEQID NOs:9-12, respectively). Its light chain variable region and lightchain CDRs, CDRL1, CDRL2 and CDRL3 having the amino acid sequences ofSEQ ID NOs:74, 76, 78 and 80, respectively, in US2015/0037339 (presentSEQ ID NOs:13-16, respectively). This antibody inhibits binding ofActivin A to ACRV2A and/or ACVR2B and inhibits signal transductionthrough one or both of these receptors. Other antibodies competing withH4H10430P for binding to Activin A or binding to the same epitope onActivin A as H4H10430P and sharing its property of inhibiting Activin Abinding to and signal transduction through ACVR2A and ACVR2B are alsoincluded.

Another exemplary antibody for use in the present methods is theantibodies designated A1 in U.S. Pat. No. 8,309,082, which ischaracterized by light and heavy chain variable regions having thesequences SEQ ID NOs:9 and 10 in U.S. Pat. No. 8,309,082 (present SEQ IDNOs:17 and 18, respectively). Its light chain CDRs, CDRL1, CDRL2 andCDRL3 having the sequences SEQ ID NO:11, 12, and 13, respectively, inU.S. Pat. No. 8,309,082 (present SEQ ID NOs:19-21, respectively), andits heavy chain CDRs, CDRH1, CDRH2 and CDRH3 having the sequences SEQ IDNOs: 62, 63 and 64, respectively, in U.S. Pat. No. 8,309,082 (presentSEQ ID NOs:22-24, respectively). Other antibodies competing withH4H10430P for binding to Activin A or binding to the same epitope onActivin A as H4H10430P and sharing its property of inhibiting Activin Abinding to and transducing a signal through ACVR2A and/or ACVR2B arealso included.

Other antibodies can be obtained by mutagenesis of cDNA encoding theheavy and light chains of any of the above-mentioned antibodies.Monoclonal antibodies that are at least 90%, 95% or 99% identical to anyof the above-mentioned antibodies in amino acid sequence of the matureheavy and/or light chain variable regions and maintain its functionalproperties, and/or which differ from the respective antibody by a smallnumber of functionally inconsequential amino acid substitutions (e.g.,conservative substitutions), deletions, or insertions are also includedin the invention. Monoclonal antibodies having at least 1, 2, 3, 4, 5and preferably all six CDR(s) that are 90%, 95%, 99% or 100% identicalto corresponding CDRs of any of the exemplified antibodies are alsoincluded. CDRs are preferably as defined by Kabat, but can be defined byany conventional alternative definition, such as Chothia, compositeKabat-Chothia, the contact definition or AbM definition (see world wideweb bioinf.org.uk/abs).

E. Small Molecule Antagonists

Antagonists of ACVR1, ACVR2A and ACVR2B can also be small moleculeantagonists. Such small molecule antagonists can inhibit an activity ofACVR1, ACVR2A, ACVR2B or Activin A. Small molecule antagonists of ACVR1include, for example, LDN-212854 described in Mohedas et al., (2013) ACSChem. Biol. 8:1291-1302, which is incorporated by reference herein inits entirety.

IV. Screening Assays

The activity of the various ACVR1, ACVR2A and/or ACVR2B antagonists andvariants or fragments thereof provided herein can be screened in avariety of assays. For example, ACVR1, ACVR2A and/or ACVR2B antagonistsand variants thereof can be screened for their ability to bind toligands or bind to ACVR1, ACVR2A or ACVR2B receptors, for their abilityto inhibit binding of a ligand to an ACVR1 and/or ACVR2 polypeptide,and/or for their ability to inhibit activity of the ACVR1 or ACVR2receptors.

The activity of an ACVR1 or an ACVR2 antagonist or variants or fragmentsthereof can be tested in vitro or in cell based assays. In vitro bindingassays and assays to measure inhibition of receptor activity are wellknown. Various assays to measure the activity of an ACVR1, ACVR2A orACVR2B antagonist are described in detail, for example, in U.S. Pat. No.7,842,663 which is incorporated by reference herein in its entirety.

The ability of the antagonist to modulate complex formation between theACVR1 or ACVR2 polypeptide and its binding protein can be detected by avariety of techniques. For instance, modulation of the formation ofcomplexes can be quantitated using, for example, detectably labeledproteins such as radiolabeled ³²P, ³⁵S, ¹⁴C or ³H), fluorescentlylabeled (e.g., FITC), or enzymatically labeled ACVR1 or ACVR2polypeptide or its binding protein, by immunoassay, or bychromatographic detection.

The ability of the ACVR1 or ACVR2 antagonist to inhibit ACVR1 or ACVR2receptor-mediated signaling can be monitored. For example, the effectsof downstream signaling such as Smad activation can be monitored using aSmad-responsive reporter gene.

ACVR1 and/or ACVR2 antagonists and variants or fragments thereof canalso be screened for activity in an in vivo assay. For example, ACVR1 orACVR2 antagonists or variants thereof can be screened for their abilityto treat FOP in a mouse model of FOP (e.g., ability to decrease ectopicbone formation). Transgenic knock-in mice have been developed that carrya conditional allele encoding Acvr1[R206H]. These Acvr1^([R206H]COIN/+)mice are described in U.S. Ser. No. 14/207,320 and PCT/US2014/026582,which are incorporated by reference herein in its entirety. This alleleexpresses the R206H variant only after activation by Cre recombinase.This allows Cre-dependent activation of Acvr1[R206H] expression atspecific tissues and at specific time by using different types of Credriver lines. In this manner the resulting mice also bypass theperinatal lethality that has been observed with a non-regulated knock-inallele of Acvr1[R206H]. Activation of Acvr1[R206H] expression in youngor in adult mice results in ectopic bone formation. For example,Acvr1^([R206H]COIN/+);Gt(ROSA26)Sor^(CreERt2/+) mice (wherein CreERt2 isa tamoxifen-regulatable recombinase (see Feil et al. (1997) BiochemBiophys Res Commun. 237(3):752-7) that has been introduced into theGt(ROSA26)Sor locus, and hence it is constitutively and globallyexpressed) develop FOP after exposure to tamoxifen. Briefly, in theabsence of tamoxifen, CreERt2 is inactive. Tamoxifen activatesexpression of Cre which then acts upon the Acvr1^([R206H]COIN/+) toconvert it to Acvr1^([R206H]/+), thereby converting the genotype of themice to mirror the genotype of the FOP patients that are ACVR1[R206H].The Acvr1^([R206H]) allele expresses Acvr1[R206H], and that is adequateto drive the development of FOP in theAcvr1^([R206H]/+);Gt(ROSA26)Sor^(CreERt2/+) mice. This bypasses theembryonic lethality experienced with conventional Acvr1^([R206H])knock-in mice, Acvr1^(tm1Emsh)(http://www.informatics.jax.org/allele/key/828153). After tamoxifentreatment, the ACVR1, ACVR2A and/or ACVR2B antagonists or a control canbe administered to the Acvr1^([R206H]COIN/+);Gt(ROSA26)Sor^(CreERt2/+)mice and the animals monitored for ectopic bone formation. SeeChakkalakal S A, et al. (20120 An Acvr1 R206H knock-in mouse hasfibrodysplasia ossificans progressiva. J Bone Miner Res. 27(8):1746-56.This assay is described in detail in the Examples below.

V. Fibrodysplasia Ossificans Progressiva (FOP)

FOP is a rare heritable disorder in which heterotopic ossification formshistologically and biomechanically ‘normal’ bone at extraskeletal sites,such as connective tissue. This disorder, although episodic, iscumulative, and results in permanent disability of increasing severity.

FOP's worldwide prevalence is approximately 1/2,000,000. There is noethnic, racial, gender, or geographic predilection to FOP. It is notonly an extremely disabling disease but also a condition of considerablyshortened lifespan.

Characteristics of FOP include, for example, congenital malformations ofthe great toe, flare-ups characterized by painful soft tissue swellingson the head, neck, and/or back with inflammation and progressiveformation of heterotopic bone via endochondral ossification.

FOP can be suspected clinically based on the presence of malformationsof the great toe. Diagnostic tests, such as x-rays or bone scan cansubstantiate great toe abnormalities and confirm the presence ofheterotopic ossification. A FOP diagnosis can also be confirmed bygenetic testing, for example, by detecting the 617 G-to-A (R206H)mutation in the ACVR1 gene.

It is common for FOP to be misdiagnosed as several other disorders,including other conditions of heterotopic ossification. FOP should bedistinguished by a differential diagnosis from disorders including, forexample, isolated congenital malformations, lymphedema, soft tissuesarcoma, desmoid tumors, aggressive juvenile fibromatosis, juvenilebunions, isolated brachydactyly, progressive osseous heteroplasia andheterotopic ossification. The presence of great toe congenitalmalformations and the painful soft-tissue flare-ups can be used todifferentiate FOP from other disorders.

Patients with FOP have congenital malformations of the great toe butotherwise appear normal at birth. The flare-ups associated with FOPstart during the first decade of life. Flare-ups can be triggered by,for example, soft tissue injury, falls, fatigue, viral infections orintramuscular injections. The result of the flare-ups is atransformation of soft tissue, such as ligaments, skeletal muscle ortendons into heterotopic bone.

There was no previous therapeutic treatment for FOP. FOP was managed bypreventative measures, such as improved safety and strategies tominimize injury, avoiding intramuscular injections and taking care whenreceiving dental care. High dose corticosteroid treatments startedwithin the first 24 hours of a flare-up can help reduce the inflammationand edema associated with flare-ups. Surgical strategies to remove theheterotopic bone are not recommended as it is counterproductive andcauses new trauma-induced heterotopic ossification.

FOP is caused by mutations in ACVR1 (also known as ALK2) that appear todestabilize the interaction of the GS domain with an inhibitorymolecule, FKBP12 (Groppe, J., et al. 2011, Cells Tissues Organs,194:291-295). FKBP12 is a negative modulator of ACVR1 and functions tostabilize the receptor in an inactive conformation (Huse, M., et al.1999, Cell, 96:425-436). See Kaplan, F. S., et al. 2012, Disease Models& Mechanisms, 5:756-762).

An example of a mutation in ACVR1 that is associated with FOP is anArginine 206 to Histidine (R206H) mutation in the intracellular domain.

A subject at risk of developing FOP includes any subject with the ACVR1R206H mutation or other mutation associated with FOP, a subject bornwith malformations of the great toe, or a subject that has a familyhistory of FOP, who has not yet developed symptoms of FOP sufficient fora diagnosis of FOP to be made by art-recognized criteria.

VI. Methods of Treatment

Methods of treating FOP, comprising administering to a subject havingFOP an effective regime of an ACVR1, ACVR2A and/or an ACVR2B antagonistare provided herein. In one embodiment, an effective regime of an ACVR2Aantagonist and an ACVR2B antagonist is administered. In a furtherembodiment, the ACVR2A antagonist is an Fc fusion protein and the ACVR2Bantagonist is an Fc fusion protein. In another embodiment, FOP istreated by administering an effective regime of an antibody againstActivin A.

“Treating” a subject with FOP means administration of an effectiveregime of an ACVR1, an ACVR2A and/or an ACVR2B antagonist, or anantibody against Activin A, to a subject that has FOP, where the purposeis to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the condition of one or more symptoms of FOP.

A “subject” is any animal (i.e., mammals) such as, humans, primates,rodents, such as mice and rats, agricultural and domesticated animalssuch as, dogs, cats, cattle, horses, pigs, sheep, and the like, in whichone desires to treat FOP. In any of the present methods, the subject canbe mammal and preferably human.

An effective regime of an Activin A, ACVR1, ACVR2A and/or an ACVR2Bantagonist, or an antibody against Activin A, means a combination ofdose, frequency and route of administration of an antagonist whichbrings a positive response in at least one sign or symptom of FOP. Apositive response can include reducing, eliminating, ameliorating,inhibiting worsening of, or delaying at least one sign or symptom ofFOP. Signs or symptoms of FOP that can be subject of a positive responseinclude for example, ectopic or heterotopic bone formation, FOPflare-ups, or pain and swelling associated with flare-ups. The regimecan be assessed in a single patient by comparing signs and symptomsbefore and after treatment. A regime is considered effective if at leastone sign or symptom gives a positive response following treatment. Aregime can alternatively or additionally be assessed by comparing signsand symptoms of population of subjects treated with an antagonist orantagonists of the present invention with a control population ofsubjects not receiving treatment. The subjects for such comparison canbe an animal model, or human subjects in a clinical trial (e.g., phaseI, phase II, IIa, IIb, or III). A regime is considered effective ifthere is a statistically significant positive response between thepopulations in at least one sign or symptom.

In some methods for treating FOP, the subject does not have and is notat risk of other conditions treatable with antagonists against ACVR1,ACVR2A, and/or ACVR2B, or an antibody against Activin A. For example,the subject can be free of any or all of type II diabetes, musculardystrophy, amyotrophic lateral sclerosis (ALS) and osteoporosis.

A. Methods of Administration

ACVR1, ACVR2A and/or ACVR2B antagonists, or an antibody against ActivinA, are usually administered directly as proteins or small molecules, butin the case of proteins can also be administered as nucleic acidencoding such proteins. Such antagonists can be administered by variousmethods, such as cellular transfection, gene therapy, directadministration with a delivery vehicle or pharmaceutically acceptablecarrier, indirect delivery by providing recombinant cells comprising anucleic acid encoding an ACVR1, ACVR2A and/or ACVR2B antagonist, or anantibody against Activin A, provided herein.

Various delivery systems can be used to administer the ACVR1, ACVR2Aand/or ACVR2B antagonists, or an antibody against Activin A, providedherein, e.g., encapsulation in liposomes, microparticles, microcapsules,recombinant cells capable of expressing the compound, receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432),construction of a nucleic acid as part of a retroviral or other vector,etc.

Methods of administration can be enteral or parenteral and includeintradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,pulmonary, intranasal, intraocular, epidural, and oral routes. Thecompounds can be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and can be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it can bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectioncan be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Omcana reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

The pharmaceutical compositions of the invention can be administeredlocally to the area in need of treatment; this can be achieved, forexample, by local infusion during surgery, topical application, e.g., byinjection, by means of a catheter, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, fibers, or commercial skinsubstitutes.

In another embodiment, the active agent can be delivered in a vesicle,in particular a liposome (see Langer (1990) Science 249:1527-1533). Inanother embodiment, the active agent can be delivered in a controlledrelease system. In one embodiment, a pump can be used (see Langer (1990)supra). In another embodiment, polymeric materials can be used (seeHoward et al. (1989) J. Neurosurg. 71:105). In another embodiment wherethe active agent of the invention is a nucleic acid encoding a protein,the nucleic acid can be administered in vivo to promote expression ofits encoded protein, by constructing it as part of an appropriatenucleic acid expression vector and administering it so that it becomesintracellular, e.g., by use of a retroviral vector (see, for example,U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment, or coating with lipids or cell-surfacereceptors or transfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see e.g.,Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination.

B. Combination Therapies

The ACVR1, ACVR2A and ACVR2B antagonists, or an antibody against ActivinA, provided herein can be administered in combination with one anotheror other treatments. In one embodiment, the method of treating FOPinvolves co-administration of an ACVR2A antagonist and an ACVR2Bantagonist. In another embodiment, the method of treating FOP involvesco-administration of an ACVR1, an ACVR2A and an ACVR2B antagonist. Inother embodiments, an ACVR1 antagonist can be co-administered with anACVR2A and/or an ACVR2B antagonist. The ACVR1, ACVR2A and ACVR2Bantagonists can be administered as separate pharmaceutical compositionsor can be administered as a single pharmaceutical composition comprisinga combination of these agents. The ACVR1, ACVR2A and/or ACVR2Bantagonists, or an antibody against Activin A, either alone or incombination, can be administered in conjunction with one or moreadditional therapeutic compounds. The combination therapy can encompasssimultaneous or alternating administration. In addition, the combinationcan encompass acute or chronic administration.

C. Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising an Activin A, ACVR1, ACVR2A and/or an ACVR2B antagonist, oran antibody against Activin A, provided herein and a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents.

These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. When necessary, thecomposition can also include a solubilizing agent and a local anestheticsuch as lidocaine to ease pain at the site of the injection. When thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. When the composition is administered by injection, an ampoule ofsterile water for injection or saline can be provided so that theingredients can be mixed prior to administration.

The Activin A, ACVR1, ACVR2A and/or an ACVR2B antagonists, or anantibody against Activin A, provided herein can be formulated as neutralor salt forms. Pharmaceutically acceptable salts include those formedwith free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, and the like, and thoseformed with free carboxyl groups such as those derived from sodium,potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The amount and frequency of the Activin A, ACVR1, ACVR2A and/or ACVR2Bantagonist, or an antibody against Activin A, administered by aspecified route effective in the treatment of FOP (e.g., effectiveregime) can be determined by standard clinical techniques based on thepresent description. In addition, in vitro assays can be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation also depends on the route of administration, and theseriousness of the condition, and should be decided according to thejudgment of the practitioner and each subject's circumstances. However,suitable dosage ranges for parenteral administration, preferablyintravenous or subcutaneous, are generally about 20-50000 micrograms ofactive compound per kilogram body weight. For antibodies to Activin Asuitable dosage ranges include 1-25 mg/kg, 2-20 mg/kg 5-15 mg/kg, 8-12mg/kg and 10 mg/kg.

Suitable dosage ranges for intranasal administration are generally about0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses can beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

Frequencies of administration also vary depending on the severity of thecondition and half-life of the agent among other factors, but aretypically between daily and quarterly, including for example, twice aweek, weekly, fortnightly, monthly, bimonthly. Agents can also beadministered at irregular intervals responsive to the patient'scondition or reduction in serum level of the agent below a thresholdamong other factors.

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise.

EXAMPLES Example 1: Use of ACVR2A-Fc/ACVR2B-Fc to Suppress Ectopic BoneFormation in a Mouse Model of FOP

Acvr1^([R206H]COIN/+); Gt(ROSA26)Sor^(CreERt2/+) were protected fromectopic bone formation after tamoxifen treatment by ACVR2A-Fc/ACVR2B-Fctreatment.

A mouse model of FOP, referred to as Acvr1^([R206H]COIN/+);Gt(ROSA26)Sor^(CreERt2/+) were given tamoxifen at 1 mg/mouse dose i.p.for eight days. Eleven mice were treated with 10 mg/kg of ACVR2A-Fc and10 mg/kg of ACVR2B-Fc twice weekly and ten mice were treated with 10mg/kg control mFc twice weekly for 6 weeks. Mice were monitored using invivo μCT at baseline, 2, 4 and 6 weeks post initiation of tamoxifenadministration. After 6 weeks, 9 out of 10 mice in the mFc group haddeveloped ectopic bone in at least one location, in contrast only 2 outof 11 mice in the ACVR2A-Fc and ACVR2B-Fc group showed development ofectopic bone and this bone was small in size. These results are shown inFIG. 1.

Example 2: Use of an ACVR1 Kinase Small Molecule Inhibitor to SuppressEctopic Bone Formation in a Mouse Model of FOP

Acvr1^([R206H]COIN/+); Gt(ROSA26)Sor^(CreERt2/+) were protected fromectopic bone formation after tamoxifen treatment by ACVR1 kinaseinhibitor LDN-212854 treatment.

16 Acvr1^([R206H]COIN/+); Gt(ROSA26)Sor^(CreERt2/+) mice were giventamoxifen at 1 mg/mouse dose i.p. for eight days. Eight mice weretreated with 3 mg/kg of the ACVR1 kinase inhibitor LDN-212854 (Mohedaset al. (2013) ACS Chem. Biol. 8:1291-1302) twice daily for 4 weeks.Eight mice were treated with vehicle control twice daily for 4 weeks.Mice were monitored using in vivo μCT at baseline, 2 and 4 weeks postinitiation of tamoxifen administration. After 4 weeks 8 out of 8 mice inthe vehicle control group showed ectopic bone formation, in 6 of thesemice the ectopic bone lesions were large in size. In contrast, in theLDN-212854 treated group, 3 out of 8 mice showed ectopic bone formation,the size of the ectopic bone lesions formed in the 3 mice were smallcompared to the vehicle control group. These results are shown in FIG.2.

Example 3: Use of an Antibody Against Activin a to Suppress Ectopic BoneFormation in a Mouse Model of FOP

23 Acvr1^([R206H]COIN/+); Gt(ROSA26)^(SorCreERt2/+) mice were treatedwith tamoxifen at 1 mg/mouse dose i.p. for eight days. Seven mice weretreated with 25 mg/kg isotype control antibody twice weekly, eight micewere treated with 25 mg/kg of Activin A antibody (H4H10446P) twiceweekly, and eight mice were treated with 10 mg/kg of ACVR2a-Fc twiceweekly for 3 weeks. Treatments with these agents were started concurrentwith initiating tamoxifen treatment. Mice were monitored using in vivomicro computer tomography (μCT) at baseline, 2 and 3 weeks postinitiation of tamoxifen administration. FIG. 3 shows that after 3 weeks,all mice in the isotype control antibody group had developed ectopicbone in at least one location, in contrast none of the mice in theActivin A antibody group showed development of ectopic bone at thistime. Two mice in the ACVR2a-Fc group developed ectopic bone at 3 weeks.

Example 4

Acvr1[R206H]COIN/+; Gt(ROSA26)SorCreERt2/+ were protected from ectopicbone formation after tamoxifen treatment by both an Activin A and anAcvr2a and b blocking antibody.

26 Acvr1[R206H]COIN/+; Gt(ROSA26)SorCreERt2/+ mice were given withtamoxifen at a 40 mg/kg dose i.p. for eight days. Eight mice weretreated with 10 mg/kg isotype control antibody (REGN1945), nine micewere treated with 10 mg/kg of Activin A antibody (H4H10446P) (REGN2477)and nine mice were treated with 10 mg/kg of an Acvr2a/Acvr2b antibodytwice weekly for 6 weeks. Mice were monitored using in vivo μCT atbaseline, 2, 3 and 4 weeks post initiation of tamoxifen administration.FIG. 4 shows that after 4 weeks, 7 out of 8 mice in the isotype controlantibody group had developed ectopic bone in at least one location, incontrast only one of the mice in the Activin A antibody treated groupand three of the mice in the Acvr2a/Acvr2b antibody treated groupdeveloped ectopic bone at 4 weeks. The size of the ectopic bone thatformed in the antibody treated group was smaller than the isotypecontrol treated group.

Example 5

Acvr1[R206H]COIN/+; Gt(ROSA26)SorCreERt2/+ were protected from ectopicbone formation after tamoxifen treatment by an Activin A blockingantibody.

35 Acvr1[R206H]COIN/+; Gt(ROSA26)SorCreERt2/+ mice were given withtamoxifen at 40 mg/kg i.p. for eight days. Eight mice were treated with25 mg/kg isotype control antibody (REGN1945), nine mice were treatedwith 25 mg/kg of Activin A antibody (H4H10446P) (REGN2477), nine micewere treated with 10 mg/kg of Activin A antibody (REGN2477) and ninemice were treated with 1 mg/kg of Activin A antibody (REGN2477) weeklyfor 6 weeks. Mice were monitored using in vivo μCT at baseline, 2, 3, 4and 6.5 weeks post initiation of tamoxifen administration. The volume ofectopic bone in each mouse was calculated from μCT images. FIG. 5 showsafter 4 weeks, all mice in the isotype control antibody group haddeveloped ectopic bone in at least one location, whereas only 2 miceeach of the Activin A antibody treated groups. At 6.5 weeks the averagetotal volume of ectopic bone in the isotype treated group was 65.4 mm3compared to 1.87 mm³ in the 25 mg/kg, 0.3 mm³ in the 10 mg/kg and 7.3mm³ in the 1 mg/kg Activin antibody treated groups.

1. A method of treating Fibrodysplasia Ossificans Progressiva (FOP), comprising administering to a subject having FOP an effective regime of an activin receptor type 2A (ACVR2A) and/or an activin receptor type 2B (ACVR2B) antagonist. 2-13. (canceled)
 14. A method of treating Fibrodysplasia Ossificans Progressiva (FOP), comprising administering to a subject having FOP an effective regime of an activin receptor type 1 (ACVR1) antagonist. 15-18. (canceled)
 19. A method of treating Fibrodysplasia Ossificans Progressiva (FOP), comprising administering to a subject having FOP an effective regime of an activin receptor type 2A (ACVR2A) and/or an activin receptor type 2B (ACVR2B) antagonist in combination with an activin receptor type 1 (ACVR1) antagonist. 20-23. (canceled)
 24. A method of treating Fibrodysplasia Ossificans Progressiva (FOP), comprising administering to a subject having FOP an effective regime of an antibody against Activin A.
 25. The method of claim 24, wherein the antibody competes for binding with antibody comprising the heavy and light chain variable regions of the antibody designated H4H10446P, H4H10430P or A1.
 26. The method of claim 24, wherein the antibody comprises the heavy and light chain variable regions of the antibody designated H4H10446P, H4H10430P or A1.
 27. The method of claim 24, wherein the antibody is chimeric, veneered, humanized or human antibody.
 28. The method of claim 24 wherein the antibody is an intact antibody.
 29. The method of claim 24, wherein the antibody is a human kappa IgG1 antibody.
 30. The method of claim 25, wherein the antibody is a human kappa IgG1 antibody.
 31. The method of claim 26, wherein the antibody is a human kappa IgG1 antibody.
 32. The method of claim 27, wherein the antibody is a human kappa IgG1 antibody.
 33. The method of claim 28, wherein the antibody is a human kappa IgG1 antibody. 